Variant tat proteins and methods for use thereof

ABSTRACT

Variants of the HIV-1 Tat protein exhibiting higher transcriptional activation and stronger P-TEFb binding than wild-type Tat are provided. In addition variants that can inhibit transcription activation by wild-type Tat are provided. Nucleic acid sequences encoding these variants, vectors and host cells for expression of these variants, and antibodies raised against these variants are also provided. In addition, methods for use of these variants and compositions containing these variants as research tools, as diagnostic tools and in the treatment of viral infections are provided.

This invention was supported in part by funds from the U.S. government(NIH Grant Nos. AI31802 and AI34552) and the U.S. government maytherefore have certain rights in the invention.

FIELD OF THE INVENTION

This present invention relates to new variants of the Tat protein thatexhibit higher transcriptional activation than wild-type Tat. The newvariants, referred to herein as “Super-Tats”, also bind P-TEFb morestrongly than wild-type Tat. Super-Tats of the present invention areuseful as research tools in studying viral replication, as diagnostictools for determination of viral infection, and in the treatment ofviral infections.

BACKGROUND OF THE INVENTION

The human immunodeficiency virus type 1 (HIV-1) Tat protein is a keyregulatory protein in the HIV-1 replication cycle. Wild-type Tat gene ofHIV-1 is required for production of viral RNA and viral replication. Tatinteracts with cellular transcriptional factors and cytokines, such astumor necrosis factor-alpha (TNF-alpha), and alters the expression of avariety of genes in HIV-1-infected and non-infected cells. Tat functionrequires its binding to a cellular positive transcription elongationfactor b (P-TEFb).

The presence of Tat specific cytotoxic T lymphocytes is correlated withstrong resistance to HIV infection (Allen et al. Nature 2000407(6802):386-390). Tat mediated pathogenic effects can also beneutralized by anti-Tat antibodies. Antibodies directed againstconserved regions of Tat, such as the cysteine rich and the lysine richdomains, have been shown to be particularly effective in inhibiting HIVreplication. In HIV-1-infected patients, a strong humoral immuneresponse against HIV-1 Tat protein is inversely correlated to peripheralblood viral load (Re et al. J. Clin. Virol. 2001 21(1):81-9).

Wild-type Tat also promotes lymphocyte infiltration and adhesionprimarily by its binding to VEGF receptor and its subsequentdimerization and activation (Mitola et al. Blood 1997 90(4):1365-72).This effort is primarily mediated by the basic domain of Tat.

Wild-type Tat modified to be defective in binding to TAR has been shownto be effective in inhibiting viral long terminal repeat (LTR)transactivation (Modesti et al. New Biol. 1991 3(8):759-68).

Tat has also been shown to be taken up and internalized by cells. Thus,fusion of a heterologous protein to Tat has been proposed as a means forcellular delivery of heterologous proteins in cell culture and livinganimals.

However, Tat has also been linked with multiple pathogenic effects. Forexample, numerous studies indicate a role for the HIV regulatory proteinTat in HIV-related inflammatory and neurodegenerative processes. HIV-1Tat protein has been linked to dementia associated with HIV infection.In addition, the Tat protein has been directly implicated in thepathogenesis of AIDS-related Kaposi's sarcoma. More recently, with theadvent of agents which prolong the life of HIV-infected patients,secretion of the Tat protein has been implicated in multiplecardiovascular diseases observed in these patients (Krishnaswamy et al.Cardiology in Review 2000 8(5):260-8). Thus, while Tat administrationhas multiple utilities, it also causes multiple pathogenic effects.

In the present invention, variants of Tat, referred to herein asSuper-Tats, are provided which exhibit similar but enhanced activitiesas compared to wild-type Tat. Accordingly Super-Tats of the presentinvention can be administered at lower levels in clinical applicationthereby minimizing pathogenic effects associated with Tatadministration.

SUMMARY OF THE INVENTION

An object of the present invention is to provide variants of thewild-type HIV-1 Tat protein, also referred to herein as Super-Tats,which exhibit higher transcriptional activation and stronger P-TEFbbinding than wild-type HIV-1 Tat.

Another object of the present invention is to provide nucleic acidsequences encoding Super-Tats, vectors comprising these nucleic acidsequences and host cells comprising these vectors which are capable ofencoding Super-Tats.

Another object of the present invention is to provide methods forproduction of Super-Tats. In one embodiment, Super-Tats of the presentinvention can be prepared via chemical synthesis. In another embodiment,Super-Tats can be prepared recombinantly.

Another object of the present invention is to provide methods of usingSuper-Tats in place of wild-type HIV-1 Tat in research applications, asan endothelial permeability factor, to inhibit viral transcription andto selectively activate latently infected cells.

Another object of the present invention is to provide antibodies raisedagainst a Super-Tat, antiviral vaccines comprising anti-Super-Tatantibodies and methods of using these vaccines to protect against HIVinfection and to neutralize the pathogenic effects of the Tat protein.

Another object of the present invention is to provide compositionscomprising a Super-Tat.

In one embodiment, the composition is a fusion protein comprising aSuper-Tat fused to another selected protein such as an enzyme. In thisembodiment, the Super-Tat facilitates uptake into cells of the selectedprotein fused thereto.

In another embodiment, the composition is a tagged fusion constructcomprising a Super-Tat fused to a tag useful in purifying the Super-Tat.Examples of such tags include, but are not limited to, HIS and FLAG.

In another embodiment, the composition is an HIV-1 molecular clone, anHIV virus or a virus such as a SHIV (simian/human chimera virus)comprising a Super-Tat. These compositions are useful for studying theeffect of highly active Tat on viral replication and latency.

In another embodiment, the composition is conditioned medium from cellsexpressing Super-Tat. Conditioned medium from cells expressing Super-Tathas a variety of uses including, but not limited to, purification ofSuper-Tat, growth medium for viral replication, and in diagnosticassays.

Accordingly, yet another object of the present invention is to providemethods and kits for diagnosing HIV infection in an individual via aconditioned medium from cells expressing Super-Tat.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to variants of the HIV-1 Tat protein,referred to herein as “Super-Tats”, that exhibit higher transcriptionalactivation and stronger P-TEFb binding than wild-type HIV-1 Tat. It hasnow been found that the introduction of a T23N change (threonine toasparagine at position 23) in wild-type HIV-1 (strain NL4-3) Tat,results in a more active protein. In particular, this Super-Tatexhibited 5-fold higher transcription activation and 3.5-fold strongerP-TEFb binding than wild-type Tat of the NL4-3 strain. Other amino acidresidues examined at this position such as Q, D, E, H, V, A, I, P, S hadeither deleterious or no effect on the activity of Tat. Still otheramino acid substitutions at this residue are expected to result inhighly active forms of Tat.

The present invention also relates to methods for production ofSuper-Tats. Various methods for production of the Super-Tats of thepresent invention can be used.

In one embodiment, the Super-Tats of the present invention can bechemically synthesized in accordance with well known techniques forpeptide synthesis.

In another embodiment, Super-Tats of the present invention can beprepared recombinantly in a host cell transfected with a vector capableof encoding the Super-Tat in the host cells. Various vectors forexpression of heterologous genes in host cells are well known and can beused in the instant invention. For example, GST-Tat T23N vectors(encoding amino acid 1 through 72 and amino acid 1 through 48) have beenproduced for expression of Super-Tat in bacteria. RSV-Tat T23N (aa 1-72and aa 1-48) vectors have also been produced for expression of Super-Tatin mammalian cells and for use in an in vitro coupled transcription andtranslation reaction. CMV-Tat-HA vectors have also been made forevaluation of expression of Tat in mammalian cells as well as forpurification. Further, as will be understood by those of skill in theart upon reading this disclosure, various other vectors and host cellssystems can also be used for recombinant expression of the Super-Tats ofthe present invention.

Thus, the present invention also relates to nucleic acid sequencesencoding a Super-Tat of the present invention and vectors comprisingsuch nucleic acid sequences. In addition, the present invention relatesto host cells comprising these vectors which are capable of expressingSuper-Tats of the present invention.

Super-Tats of the present invention are expected to be useful ininhibition of HIV replication. Tat function requires binding to TAR, anRNA element in the viral LTR that is synthesized in early in thetranscription process. Thus, in one embodiment, Super-Tats of thepresent invention have been made that are defective in binding to TARbut still retain their ability to bind to P-TEFb. One variant, referredto herein as Super-TatK7, was produced by changing five arginines in thebasic domain of Super Tat to lysines. Co-expression of this protein incells along with wild-type Tat in LTR transactivation assays inhibitedreporter gene expression 6.5 fold. Expression of this protein in cellsinfected with virus is expected to inhibit viral transcription bycompeting with viral Tat for binding to P-TEFb. It is believed thatSuper-Tat defective in TAR binding will be a more potent inhibitor ofviral replication than wild-type Tat modified to be defective in bindingto TAR as described by Modesti et al. (New Biol. 1991 3(8):759-68).Accordingly, the Super-Tats of the present invention can be used as anadditional anti-viral compound to an existing anti-HIV drug regimen.

The Super-Tats of the present invention may also function as effectiveimmunogens for raising anti-Tat antibodies. Antibodies against theSuper-Tats of the present invention can be raised in accordance withwell known techniques. These antibodies can then be incorporated intovaccines and administered to individuals to protect against HIVinfection and to neutralize Tat mediated pathogenic effects inindividuals already infected with HIV. Techniques for preparation ofvaccines from antibodies are also well known.

In addition, the Super-Tats of the present invention may have a moreaccessible basic domain due to their unique conformation. Accordingly,the Super-Tats may activate the VEGF receptor more potently and may beuseful as endothelial permeability factors.

As demonstrated herein, Super-Tats of the present invention interactmore strongly with P-TEFb, particularly with its cyclin T1 subunit.Accordingly, the Super-Tats of the present invention can also be used inresearch applications in place of wild-type Tat. For example, toinvestigate the requirement for P-TEFb in transcription, nuclearextracts are depleted of endogenous P-TEFb by incubating them with a HIVTat column. Purified P-TEFb can then be added back to the depletedextract in controlled amounts or at set intervals to investigate itsrole in transcriptional activity. A Super-Tat of the present inventioncan be used instead of wild-type Tat to more efficiently remove P-TEFbfrom these extracts. Further, lower amounts of the Super-Tat as comparedto wild-type Tat can be used to achieve the same effect.

Use of anti-viral compounds to treat HIV fails to completely eradicateHIV infection (Siliciano, J. D. and Siliciano, R. F. J. Clin. Invest.2000 106(7):823-5). This is believed to be due to the persistence oflymphocytes containing stably integrated, yet transcriptionally silent,proviruses. Occasional reactivation of these lymphocytes continues toprovide a source of infection. It is believed that Super-Tat mayselectively activate viral replication in resting cells and result inthe enhanced clearance of these cells from the patients system.Alternatively Super-TatK7 may be utilized to further augmentanti-retroviral therapy and enhance suppression of viral reactivation.Accordingly, treatment of patients with doses of Super-Tat orSuper-TatK7, along with other anti-viral compounds is expected to bebeneficial.

The present invention also relates to various compositions comprising aSuper-Tat and their use in a variety of applications.

For example, chemically synthesized Super-Tats can be added to cellmedia for uptake by cells and subsequent viral activation. Chemicallysynthesized forms of this peptide are also useful for structural studiessuch as via 2-dimensional nuclear magnetic resonance (NMR) spectroscopy.

Tagged fusion constructs such as (multiple Histidine-tagged (HIS-tagged)or Hemagglutanin-tagged (HA-tagged) constructs, are useful primarily forproduction in cells followed by purification over an affinity column.The HA fusion construct of Super Tat is already known to be active intransactivation assays. Proteins expressed in cells often containpost-translational modifications including, but not limited to,acetylation, myristoylation and glycosylation, required for full in vivofunction. The present invention also includes post-translationalmodified versions of Super-Tat.

Viral vectors containing Super-Tat are useful in studies of Tattransactivation and viral replication. Super-Tat can be incorporatedinto viral vectors including, but not limited to, adenovirus andretrovirus vectors in accordance with well known techniques.

Viral particles of HIV viruses and SHIV, a simian/human chimera virus,comprising Super-Tat of the present invention are useful for studyingthe effect of highly active Tat on viral replication and latency.

Stably transfected cell lines, such as mammalian cells stablytransfected with Super-Tat, can be used for infection of various virusstrains and to investigate the role of Super-Tat in the viral lifecycle. Additionally, stably transfected cells can be used to propagateselected HIV viral strains that do not grow well under other conditions.

Conditioned medium from cells expressing Super-Tat can also be prepared.Animal cells expressing Tat normally release some amount of Tat into themedium. Thus, it is expected that cells expressing Super-Tat will alsorelease some amount into the medium. This “conditioned” medium has manyuses. For example, the conditioned medium can be used to purifySuper-Tat. The conditioned medium can also be used as a growth mediumfor viral replication. HIV culture is often used as part of a panel oftests to confirm if an individual is infected. Use of the Super-Tatconditioned media is expected to enhance the sensitivity of these testsby decreasing the number of days required to detect viral replication.

Accordingly, the present invention also relates to the use of Super-Tat,and in particular, conditioned medium comprising Super-Tat in assays andkits for diagnosis of HIV infection in patients.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1 Production of a Super-Tat

The Tat gene from the HIV-NL4-3 molecular clone was subcloned into pUC19vector (Yamisch et al. Gene 1985 33:103-119) after digestion with Bam HIand Sal I restriction enzymes. Mutagenic PCR, using the QuickchangeSite-Directed Mutagenesis Kit (Stratagene, Calif.) was carried out usingthe primer pair CTG CTT GTA ACA ATT GCT AT (SEQ ID NO:1)/TTG TTA CAG CAGTTT TAG G (SEQ ID NO:2) to change the threonine at amino acid position23 to asparagine. The sequence of the resulting Tat protein isMEPVDPRLEPWKHPGSQPKTACN(SEQ IDNO:3)\NCYCKKCCFHCQVCFITKALGISYGRKKRRQRRRAHQNSQTHQASLSKQ (SEQ ID NO:4).This construct is called pUC-Tat T23N.

Example 2 Measurement of Transcriptional Activity

A glutathione-S transferase tagged version of Tat T23N (1-72) was madeby subcloning into the pGEX2T vector (Invitrogen). GST-Tat wild type(NL4-3) was also constructed. A Tat-associated kinase assay was carriedout using U937 cell extract (Ramanathan et al. J. Virol. 199973(7):5448-58). Briefly, U937 cells were treated with 5 ng/ml PMA(Sigma-Aldrich, MO) to induce P-TEFB. Cells were harvested and proteinextracts prepared. The GST-Tat proteins were expressed in E. coli andbound to Glutathione-Sepharose beads (Amersham-Pharmacia, Sweden),washed several times and incubated with 400 micrograms of U937 cellextract to pull-down the endogenous P-TEFb complex. The beads were thenwashed again and a kinase reaction was performed with a synthetic CTDsubstrate and radiolabeled ATP. The reaction products were resolved on aSDS-polyacrylamide gel and the extent of CTD phosphorylation quantifiedon a Phosphoimager (Molecular Dynamics, Sweden). This experiment wasalso conducted using extracts of 293 cells. With either cell extract theP-TEFb complex bound to GST-Tat T23N phosphorylated CTD peptide 3.5-foldmore than complex bound to wild-type Tat. The increased binding ofGST-Tat T23N to endogenous P-TEFb was confirmed by immunoblotting todetect CDK9, the kinase subunit of P-TEFb.

Transcriptional transactivation of the HIV-1 LTR was measured byco-transfection experiments. The Tat gene from pUC-Tat T23N wassubcloned into an RSV-driven expression vector pRc-RSV (Invitrogen, CA).U937 cells were electroporated with 4 μmicrograms of HIV-LTR-Luciferase,7 micrograms of RSV-renilla Luciferase and 0.5 micrograms of RSV-TatT23N or RSV-Tat wild-type (NL4-3). Cells (1×10⁶) were electroporated in250 microliter of RPMI 1640 medium at 250 V, 1100 micro F and 186 Ohmresistance in 4 mm gap electroporation cuvettes. Cells were cultured in10 ml growth medium (RPMI 1640+100% FBS) for 12 hours and harvested.Cell pellets were lysed in 50 microliter of Promega Lysis Buffer and 20microliter was used in a Dual Luciferase Assay (Promega, WI). Thetransfection efficiency was normalized to renilla luciferase activity.In this assay Tat T23N transactivated 3-fold more than wild-type Tat.

1. A variant of wild-type HIV-1 Tat protein which exhibits highertranscriptional activation and stronger P-TEFb binding than wild-typeHIV-1 Tat.
 2. The variant of claim 1 comprising a mutation at position23 of wild-type HIV-1 Tat.
 3. The variant of claim 2 wherein themutation at position 23 is a change from threonine to asparagine oranother amino acid that results in higher activity.
 4. A nucleic acidsequence encoding the variant of claim
 1. 5. A vector comprising thenucleic acid sequence of claim
 4. 6. A host cell comprising the vectorof claim
 5. 7. A method of inhibiting viral transcription in a cellcomprising contacting the cells the variant of claim
 1. 8. A method ofselectively activating cells latently infected with HIV comprisingcontacting the cells with the variant of claim
 1. 9. An antibody raisedagainst the variant of claim
 1. 10. A vaccine comprising the antibody ofclaim
 9. 11. A method for protecting an individual against HIV infectioncomprising administering to the individual the vaccine of claim
 10. 12.A method of neutralizing pathogenic effects of HIV-1 Tat protein in anindividual infected with HIV-1 comprising administering to theindividual the vaccine of claim
 10. 13. A fusion protein comprising aselected protein fused to the variant of claim
 1. 14. A method forfacilitating uptake of a selected protein comprising fusing the selectedprotein to the variant of claim
 1. 15. A tagged fusion constructcomprising the variant of claim 1 fused to a tag for purification of thevariant.
 16. A defective HIV virus comprising the variant of claim 1 anda simian/human chimera virus.
 17. A conditioned cell medium obtainedfrom cells expressing the variant of claim
 1. 18. A diagnostic assay forHIV infection comprising the conditioned medium of claim
 17. 19. A kitfor diagnosis of HIV infection comprising the conditioned medium ofclaim 17.